Discovery Of Glyceraldehyde-3-phosphate Dehydrogenase From Riemerella Anatipestifer And Its Roles In Pathogenesis

Background: R.anatipestifer, a member of the familyFlavobacteriaceae in rRNA, is a Gram-negative bacterium exhibitingnon-motile, non-spore-forming, rod-shaped properties. It can infectdomestic ducks, goose, turkeys, and other wild birds. The infection todomestic ducks, known as infectious serositis, which is characterized byhigh morbidity, high mortality, can cause such symptoms as fibrinouspericarditis, perihepatitis, airsacculitis, caseous salpingitis, meningitisan.R.anatipestifer has21serotypes,16of which have been isolated andidentified in China. Among the known serotypes of R.anatipestifer, therewere huge variations of virulence between each serotype or betweendifferent strains even within same serotype. The diversity of serotype andincreasing resistance have led it to be one of the most hazard pathogens toduck-raising industry. Although there are four genome sequences of R.anatipestifer reported so far, including strains ATCC11845, RA-GD, RA-YM and RA-SG, little was known about the molecular basis of pathogenicityof R.anatipestifer infection except VapD, CAMP cohemolysin and OmpA. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), amultifunctional protein, besides its classical glycolytic roles in convertingD-glyceraldehyde-3-phosphate into1,3-diphosphoglycerate, is involved ina number of fundamental cellular pathways such as maintenance of DNAintegrity, intracellular membrane trafficking, histone gene regulation,receptor mediated cell signaling, protection of telomeric DNA,posttranscriptional gene regulation, autophagy, apoptosis and oxidativestress response, which depend on the ability of GAPDH to modify its distinctsubcellular localization. The GAPDH is highly conserved across species,and often used as model proteins or reference standard inNorthern/Western-blot and other analysis such as a gene regulatory,catalytic mechanism.What’s more, GAPDH is an important pathogenicfactor to some bacteria, fungi, and parasites, such as Trpanosoma brucei,Streptococcus, Escherichia coli, Leishmania parasite, Candida albicans,Plasmondium falciparum, Schistosoma haematobium and Mycoplasmagenitalium. It presents on the surface of cell and may facilitate theircolonization and invasion of host tissues by interacting directly with hostsoluble proteins and surface ligands. Based on its roles of pathogenesis insome pathogenic microorganisms, GAPDH has become the key object in thestudying of resistant drugs to malaria, schistosomiasis, EPEC and EHECinfection and other serious diseases endangering human health, and has greatvalue in theory and application. In the previous study, we have discovered that there is a homolog of gap gene in the genome DNA of R.anatipestifer,but there is little known on its features and coding product, and thepossibility of extracellular enzyme,the biological activity have not beenreported until now.Objects: In this study, GAPDH activity in R.anatipestifer (namedRaGAPDH) clinical isolates from Sichuan, Chongqing area was detected.The molecular characteristics of gap gene and its coding protein wereanalyzed. Besides it, we would attain the recombinant RaGAPDH withGAPDH activity, and discovery the distribution, biological activity ofRaGAPDH. The gap deleted mutants of R.anatipestifer CZ2strain would beconstructed for discovering the roles of RaGAPDH in the pathogensis ofR.anatipestifer. Thus, this study would lay the foundation for exploring themolecular basis and mechanism of disease, and furthering research the rolesof extracellular RaGAPDH on the pathogenesis of R.anatipestifer.Contents and Results:(1)The GAPDH activity presenting on the cell surface of22R.anatipestifer isolates from Sichuan, Chongqing area was detected. Theresults showed that all18strains presented GAPDH activity besides DX1,PN1209, YC1and YC2, and there are huge variations of GAPDH activitybetween each serotype or between different strains even within sameserotype. The extracellular GAPDH activity in the cell free supermant atdifferent culture time of CZ2, SC12strains which with high and low GAPDH activity respectively on the cell surface were detected. The resultsshowed that the extracellular GAPDH activity of CZ2strain was higher thanSC12strain during the course of analysis. The RT-PCR results confirmedthat the expression of RaGAPDH encoding gene of CZ2was significantlyhiger than that of SC12, consistenting with the results of GAPDH activity onthe cell surface.(2) The RaGAPDH encoding gene was cloned and sequenced bygenome walking. The gap gene (including1002bp), encodes334aminoacids; its theoretical molecular weight and PI were35947.07Da,7.0respectively. Hydrophilic analysis of the RaGAP protein did not indicate thepresence of any transmembrane spanning regions or a signal sequence.Despite considerable divergence from other characterized GAPDHmolecules, the RaGAPDH was also composed of two basic domainscommon to members of the GAPDH family of enzymes: the NAD+bindingdomain (residues4-152) and the catalytic domain (residues157-314). Aphylogenetic tree was built by using the MEGA5.2program to determine theorigin and identity of the RaGAPDH. The RaGAPDH was localized on thesame branch as enterohemorrhagic Escherichia coli (EHEC), belonging tothe class I of bacteria GAPDH. The phylogenetic tree also showed that theorigin of RaGAPDH is monophyletic.(3) The prokaryotic expression vector of gap gene of R.anatipestiferCZ2strain had been constructed successfully. The recombinant RaGAPDH with a specific activity of6U/μg could bind the plasminogen and fibrinogentogether and result in engorgement, dilation and tortuous of vasculars inliver and brain. The blood and serum biochenmical index of10-day-oldhealthy Sichuan ducklings injected with recombinant RaGAPDH throughjugular vein did not expresent difference between two groups, such as redblood cell (RBC), lymphocytes (LT), serum glucose (GLU), total protein(TP), alkaline phosphatase (AKP), alanine aminotransferase (ALT), lactatedehydrogenase (LDH), total cholesterol (TCHO) besides blood WBC whichwas significantly higher than that in the control group.(4) The BALB/c mice and healthy Sichuan ducklings were immunizedwith recombinant RaGAPDH after blended with Freud’s adjuvant in order toobtain anti-recombinant RaGAPDH serum which used in western-blotanalysis. The results of western-blot showed that there was a proein bandwith a molecular weight of29-44.3kDa among extracellular protein of AF,CZ2strain, and could react with anti-Recombinant RaGAPDH serum, whilecan not with anti-OmpA serum. The growth of R.anatipestifer CZ2straincells cultured in TSB with anti-RaGAPDH anti-recombinant RaGAPDHserum rised to6.15×103%, significantly higher than1.31×103%in controlgroup. The morphology of CZ2strain cell changed from short rods in normalconditions into spheres. The serum provided passive immune protection,whose index is up to37.5%for the ducklings infected with R.anatipestifer,and reduces clinical index significantly, while it has no significant effect on the pathogenic bacteria distribution in infected duck liver, heart and brain.(5) The pMEG-375was used to construct gap deleted mutants ofR.anatipestifer CZ2. The genome DNA of CZ2△gap mutant was used astemplate, and amplified the OmpA, Km, gap and16sRNA. The resultsshowed that the OmpA, Km could be amplified, while gap gene could not.The16sRNA sequence of CZ2△gap has a100%of homology with CZ2parent.The results of biological assay showed that there is no difference indyeing properties, morphology, and biochemical characteristics, while thegrowth rate and the invasive ability decreased significantly (0.01<P<0.05)comparing to the parent strain.Conclusion：(1) This is the first report for documenting GAPDHactivity in R.anatipestifer. The extracellular RaGAPDH of R.anatipestiferwas a homolog of GAPDH, and there were huge variations of GAPDHactivity among different R.anatipestifer isolates. The variations may beclosely related to the expression of gap gene. The gap gene ofR.anatipestifer (including1002bp), encodes334amino acids; its theoreticalmolecular weight and PI are35947.07Da,7.0respectively. Hydrophilicanalysis of the RaGAP protein does not indicate the presence of anytransmembrane spanning regions or a signal sequence. Despite considerabledivergence from other characterized GAPDH molecules, the RaGAPDH isalso composed of two basic domains common to members of the GAPDHfamily of enzymes: the NAD+binding domain (residues4-152) and the catalytic domain (residues157-314). A phylogenetic tree is built by using theMEGA5.2program to determine the origin and identity of theR.anatipestifer gap gene. The RaGAPDH is localized on the same branch asenterohemorrhagic Escherichia coli (EHEC), belonging to the class I ofbacteria GAPDH. The phylogenetic tree also showed that the origin ofRaGAPDH is monophyletic.(2) The prokaryotic expression vector of RaGAPDH encoding gene hadbeen constructed successfully and a high level of expression of recombinantRaGAPDH was attained by adding0.5mM of IPTG and incubating theculture at37℃for3h. The recombinant RaGAPDH with a specific activityof6U/μg could bind the plasminogen and fibrinogen together, and wastoxtic to tissue and organs in ducklings,which is likely to cause engorgement,dilation and tortuous of vasculars in liver and brain. The anti-recombinantRaGAPDH serum could neutralize the toxicity and provide passive immuneprotection.(3) The gap deleted mutants of R.anatipestifer CZ2had beenconstructed successfully. The CZ2△gap mutant was loss of RaGAPDH andits invasiveness to the duck embryo fibroblast decreased significantly,suggesting that RaGAPDH might be a key virulence factor in thepathogenesis of R.anatipestifer.